Tumor-suppressive microRNA-152 inhibits the proliferation of Ewing’s sarcoma cells by targeting CDK5R1

We elucidated the mechanism through which the reduced expression of miR-152 leads to the overexpression of its target cyclin-dependent kinase-5 activator 1 (CDK5R1) in Ewing’s sarcoma (ES) cells and the role of this mechanism in the proliferation of ES cells. To explore possible oncogenic factors in ES, we conducted microarray-based investigation and profiled the changes in miRNA expression and their effects on downstream mRNAs in five ES cell lines and human mesenchymal stem cells (hMSCs). miR-152 was significantly downregulated, while cyclin-dependent kinase-5 activator 1 (CDK5R1) expression was significantly upregulated in all tested ES cells as compared to hMSCs. The overexpression of CDK5R1 led to the activation of CDK5, enabling the phosphorylation of retinoblastoma protein and persistent overexpression of CCNE. Moreover, miR-152 suppressed cell proliferation via cell cycle retardation, and its upregulation reduced tumor size and CCNE expression in tumor tissues. The overexpression of cyclin E (CCNE) has been detected in ES cells, but the detailed mechanisms have not been previously elucidated. These findings identify the miR152-CDK5R1 signaling axis as a critical mechanism for tumorigenesis that may serve as a new therapeutic target in Ewing’s sarcoma. We believe that our results will aid in the development of effective treatment strategies for patients with ES.


miR-152 directly targeted the 3′-UTR of CDK5R1
Two possible binding sites of miR-152 and 3′-UTR of CDK5R1 were predicted using BLAST (Fig. 1a) and Target Scan 8.0 (https:// www.targe tscan.org/ vert_ 80/) (Supplemental Fig. S2).Oligonucleotides with sequence mutations in the seed region of miR-152 were created and transfected into ES cell lines (Fig. 1b).In comparison with the control-miR and miR-152 mutant groups, overexpression of miR-152 was observed only in the miR-152-transfected group (p < 0.01).Overexpression of miR-152 was confirmed in cells transfected with oligonucleotides (miR-152 mutant), thereby showing that the transfection was successful.To determine whether miR-152 targeted CDK5R1, we evaluated the levels of CDK5R1 mRNA.Compared with cells transfected with control-miR and miR-152 mutant, the cells transfected with oligonucleotide miR-152 mimic showed significantly reduced expression of CDK5R1 (p < 0.01; Fig. 1c).To clarify the direct target of miR-152, sites 1 and 2 of the 3′-UTR of CDK5R1 mRNA transcript were inserted separately into a dual-luciferase vector pmirGLO, and the ability of miR-152 to regulate the reporter gene was examined (Fig. 1d).Addition of miR-152 mimic resulted in significant repression of sites 1 and 2 compared to that in control-miR (100%).Further, we confirmed the direct and specific regulation of CDK5R1 3′-UTR binding site mutations (sites 1 and 2) by miR-152 mimic using a luciferase reporter assay.There were no significant differences between mutant site 1 and 2 and control-miR (Fig. 1e).
Thus, our results showed that the 3′-UTR of CDK5R1 was a target of miR-152.It was found that successfully transfected miR-152 targets mRNA.Therefore, we then examined whether miR-152 reduced the expression of the CDK5R1 protein (Fig. 1f).
The quantification of protein expression showed a reduced expression of CDK5R1 in cells transfected with 20 nM miR-152 mimic compared with the untreated cells (Fig. 1g).Protein expression after transfection with CDK5R1 siRNA was observed (Fig. 1h).The transfection with CDK5R1 siRNA resulted in reduced expression of CDK5R1 protein, after 20 nM (Fig. 1i).Thus, the abovementioned results clearly showed that miR-152 targeted CDK5R1 and reduced its protein expression.

Cleavage of CDK5R1 (p35 into p25) activate CDK5
Cleavage of CDK5R1 (p35) into p25 by calpain in the cytoplasm leads to the formation of the CDK5-p25 complex, which is then translocated into the nucleus where it is activated and increases the kinase activity 16 .To determine whether this mechanism can also be replicated in SKES-1 cells, we evaluated the dynamics of p35/ p25 and CDK5 in the cytoplasm and nuclear fractions after calpain activation and calpain inhibitor administration (Fig. 2a).In the cytosolic fraction, activation of calpain led to the cleavage of p35 into p25, which was then blocked by a calpain inhibitor.Overexpression of p25 led to the activation of CDK5, which showed that calpain cleaved p35 into p25 while inducing CDK5 overexpression in SKES-1 cell lines as well.In the nuclear fraction, calpain activation led to overexpression of p25 and absence of p35, and nuclear p25 was downregulated by a calpain inhibitor.Calpain activation and inhibition resulted in the cleavage of p35 to form p25. In SKES-1 cells, p35 was cleaved in the cytoplasm to form p25, which was subsequently translocated to the nucleus, indicating that CDK5 was activated (Fig. 2b).
Next, we transfected miR-152 or CDK5R1 siRNA to evaluate changes in the expression of p35 and subsequent changes in the activity of CDK5 (and its cofactor, p25) as a complex (Fig. 2c).We observed reduced expression of p35 and p25 and CDK5 in the cytosolic fraction (input) in the miR-152 mimic and CDK5R1-siRNA transfection group.Further, IP using CDK5 antibodies showed reduced expression of p35 in the cytosolic fraction in miR-152 mimic and CDKR1-siRNA groups (Fig. 2d).The expression of p35/p25 and CDK5 in the nuclear fraction was significantly reduced in the miR-152-CDK5R1-siRNA transfection group compared with the untreated group (Fig. 2e).
In the nuclear fraction, input showed a reduction in p35 expression in the miR-152 mimic and siRNA treatment groups, and IP with the CDK5 antibody showed a reduction in p25 expression (Fig. 2f).Our results demonstrated that reduced expression of miR-152 in all ES cells allowed the overexpression of p35/p25, which led to the activation of CDK5 in the nucleus.

miR-152 suppressed cell proliferation due to cell cycle retardation
We examined the effect of transfection with miR-152 mimic and CDK5R1 siRNA on cell proliferation.When compared to the control group, the proliferation of SKES1 cells was separately inhibited by 20 nM of miR-152 mimic and CDK5R1 siRNA.Additionally, growth changes were observed in RDES cells.Proliferation assays in SKES-1 and RDES showed a significant decrease in the miR-152 mimic (20 nM) and CDK5R1 siRNA-treated group when compared to the untreated group (Fig. 3a).An apoptosis assay was performed to examine the possible cytostatic effects caused by the induction of apoptosis (Fig. 3b).Expression of cleaved PARP and cleaved caspase 3 were unaltered in the miR-152-CDK5R1-siRNA group compared with that in the untreated group, whereas transfection with miR-152 or CDK5R1 siRNA reduced the expression of CDK5R1, thereby leading to reduced cell proliferation (Supplemental Fig. S3a).However, the induction of apoptosis did not contribute to this process at the condition in which growth inhibition was observed in the proliferation assay.Furthermore, flow cytometry analysis using Annexin V-FITC/PI double staining showed that cell apoptosis was not observed among all groups (Supplemental Fig. S3b).Treatment with miR-152 mimic and CDK5R1 knockdown did not Luciferase reporter assay for the direct and specific interaction of miR-152 with predicted two target sites in the 3′-UTR of CDK5R1.Reporter activity was significantly reduced in miR-152 mimic transfected cells.Control-miR cells were used as control here for normalization.The mutations at the two target sites did not reduce luciferase activity in response to the miR-152 mimic; it remained equivalent to the Control-miR group.Images (f) and plots (g) from the immunoblot analysis of CDK5R1 protein expression following changes in miR-152 expression.Images (h) and plots (i) of protein expression of CDK5R1 following changes in the concentration of CDK5R1 siRNA.GAPDH level was used as loading control.
Vol:.( 1234567890 www.nature.com/scientificreports/induce apoptosis (Fig. 3c).CCK-8 assays showed a decrease in cell viability following transfection with miR-152 mimic and CDK5R1 knockdown (Fig. 3d).miR-152 induction and CDK5R1 knockdown decreased the mRNA expression of Ki-67 and PCNA, indicating a reduction in cell proliferation ability (Fig. 3e).Next, a cell cycle analysis of BrdU uptake was performed (Supplemental Fig. S3c).Acceleration of the cell cycle in the G1 phase and delay in the cell cycle in the S and G2/M phases were observed in the miR-152 mimic and CDK5R1 siRNA groups compared with the untreated group (Fig. 3f).The delay in the cell cycle, and not cell death by apoptosis, caused a reduction in cell proliferation after transfection with miR-152 and knockdown of CDK5R1.The colony-formation assays indicated a considerable decrease in colony formation in SKES-1 cells following transfection with miR-152 mimic and CDK5R1 knockdown (Supplemental Fig. S3d).A statistically significant reduction of the transformation response in the presence of miR-152 mimic and CDK5R1 knockdown in comparison with both untreated and control groups (Fig. 3g).

Changes in the expression of cell cycle factors in each phase
To examine the effects of miR-152 and CDK5R1 on the cell cycle, changes in cell cycle factors were synchronized in the G0/G1, S, and G2/M phases.Expression of phosphorylated Rb (p-Rb) in the G1 phase after transfection with miR-152 mimic or CDK5R1 siRNA was significantly reduced compared with that in the untreated miRcontrol and siRNA-control groups (Fig. 4a).Expression of CCNE was significantly reduced in the miR-152 The cytoplasmic fraction was normalized using GAPDH, and the nuclear fraction was normalized using Lamin B1 as reference.In the nuclear fraction, p25 was increased by calpain activation and p35 was absent, whereas nuclear p25 was decreased by calpain inhibitor.Cytoplasmic p25 was formed from the cleavage of p35 by calpain activation and inhibition.Effects of miR-152 mimic transfection and CDK5R1 knockdown on p35/p25 and CDK5 in the cytoplasm fractions were analyzed by western blot analysis (c) and band concentration quantification (d).Immunoprecipitation (IP) was performed with CDK-5, followed by western blotting with the indicated antibodies.The Input was normalized using GAPDH, and the proteins immunoprecipitated with CDK5 were normalized to CDK5.Western blotting analysis of p35/p25 levels in SKES-1 cells transfected with miR-152 and CDK5R1 siRNA in nucleus fraction (e).Immunoprecipitation (IP) was performed with CDK-5, followed by western blotting with the indicated antibodies.Quantification (f) of changes in the protein expression of co-precipitated p35/p25 in the nuclear fractions were performed.The Input of nucleus fraction was normalized using Lamin B1, and the proteins immunoprecipitated with CDK5 were normalized to CDK5.transfection and CDK5R1 knockdown group compared with the untreated miR-control and siRNA-control groups.Consistent with the decrease in CCNE expression, p-CDK2 (Thr160) and p-CDC2 (Thr161) expression was also significantly reduced.CDK2 is actively phosphorylated and downstream Rb is phosphorylated (Ser807/811) due to higher CCNE in ES cells than in normal cells.Thus, miR-152 transduction and CDK5R1 reduction appropriately suppressed CDK2 and Rb activity (Fig. 4b).Immunofluorescence staining showed that nuclear expression of p-Rb was significantly reduced in the miR-152-transfection-CDK5R1-knockdown group compared with the untreated groups.Compared with the restriction point (R-point), the expression level of CCNE is a more significant contributor of timing the cell division, which is strictly controlled in the G1 phase (Fig. 4c).In the S phase, CCNE overexpression occurred upon increased Rb phosphorylation, even in normal cells.Both CCNA expression and CDC2 phosphorylation occurred actively; the expression of CDK2 and Rb were also reduced by miR-152 transfection and CDK5R1 knockdown (Fig. 5a).The expression of p-Rb was significantly reduced in cells transfected with miR-152 and CDK5R1 knockdown compared with cells of the untreated cells (Fig. 5b).The phosphorylation of RB and nuclear accumulation after immunofluorescence staining were significantly reduced in cells with miR-152 transfection and CDK5R1 knockdown compared with untreated cells (Fig. 5c).In the G2/M phase, the levels of CCNE and CCNA, which should be reduced, remain unchanged, and the phosphorylation of CDK2 and CDC2 is high (Fig. 6a).In ES cells, the phosphorylation of Rb continues in the G2/M phase along with the expression of target gene.These are suppressed by miR-152 transfection and CDK5R1 knockdown (Fig. 6b).Reduced expression of Rb protein phosphorylation was observed in cells with miR-152 transfection and CDK5R1 knockdown (Fig. 6c).This indicates the overexpression of CCNE in cells with reduced expression of miR-152 and overexpression of CDK5R1, even in the G2/M phase.Phosphorylation of Rb proteins in the initial G1 phase after cell division and subsequent overexpression of CCNE after cell division created conditions that allowed cells to easily cross the R-point.

Upregulated miR-152 reduced tumor size in mice and decreased CCNE expression in tumor tissues
Tumors from mice xenografted with Ewing's sarcoma cells: Control-miR, miR-152-mimic, Control-siRNA, and CDK5R1-siRNA-transfected cells, were analyzed (Fig. 7a).Significant tumor shrinkage was observed in the miR-152 mimic-CDK5R1 knockdown groups compared with the negative control groups (Fig. 7b).CDK5R1 and CCNE protein levels were significantly downregulated in tumor tissues collected from the miR-152-mimic and CDK5R1-siRNA groups (Fig. 7c and Supplemental Fig. S4a).Expression of CDK5R1 and CCNE in tumor tissues was evaluated by immunostaining (Fig. 7d).The number of CDK5R1-positive cells was significantly lower in the miR-152 and CDK5R1 knockdown group compared with the negative control group.Similarly, the number of CCNE-positive cells was significantly lower in the miR-152-CDK5R1-knockdown group than in the negative control groups (Supplemental Fig. S4b).In Ewing sarcoma cells, miR-152 was down-regulated and its target, CDK5R1, was up-regulated.Within the cytoplasm, p25 underwent cleavage by calpain, was bound to cytoplasmic CDK5, and was translocated to the nucleus.Rb www.nature.com/scientificreports/protein was phosphorylated by the activated CDK5, and CCNE expression was upregulated, thus leading to CCNA accumulating as a positive loop and resulting in abnormal cell-cycle progression (Fig. 7e).

Discussion
ES is a highly malignant sarcoma that is commonly observed in young adults and mainly caused by the fusion gene EWSR1-Fli1, which is a member of the ETS family 17 .In this study, we demonstrated the relationship between abnormalities in the expression of miR-152, one of the tumors suppressive miR, and its target gene CDK5R1 in ES.Our previous findings suggested the involvement of CCNE in the proliferation of ES cells 18 , and whole genome analysis of the cDNA array and cell cycle-related factors demonstrated the overexpression of CDK5R1 in all ES cell lines, thereby aiding in elucidating the underlying mechanism.CDK5R1, known as p35, activates CDK5 serine/threonine kinase by binding to CDK5 19 .Calpain protein cleaves p35 into p25, forming a complex with CDK5, which is then translocated into the nucleus to promote cell proliferation through Rb phosphorylation 20 .CDK5, which is mainly expressed in nerve cells, plays an important role in neural tissues (e.g.controlling the function of the central nervous system, synaptogenesis, etc.) 21,22 and is involved in cell proliferation through CDK5 upregulation in malignant cells 10 .We demonstrated that miR-152 targeted CDK5R1 at the mRNA and protein expression levels.Further, we showed that following reduced miR-152 expression, calpain cleaved the CDK5-p35 complex into CDK5-p25, which was then translocated into the nucleus and activated; this is consistent with previous studies.Calpain stimulation did not induce changes in nuclear p25 levels significantly.It is possible that in Ewing sarcoma cell lines, the persistent high expression of CDK5R1 leads to a state of high endogenous p25 production.Therefore, even with external Calpain stimulation, it may not have resulted in a more significant difference.On the contrary, the decrease in nuclear p25 upon Calpain inhibition suggests the validation of the cleavage of p35 into p25 by Calpain.The reduced expression of CDK5R1 after transfection with miR-152 mimic or CDK5R1 siRNA showed cytostatic effects via a delay in the cell cycle.Previous studies have also reported CDK5-mediated upregulation of the cell cycle and tumorigenesis 9 .Due to the reduced expression of CDK5R1, reduced phosphorylation of Rb proteins and reduced expression of E2F1 and CCNE in the G1 phase.In the G1 phase, E2F1 is usually inhibited by Rb if the cells pass through the R-point without phosphorylation of Rb proteins 23 .In ES cells, overexpression of cellular CCNE from the G1 phase did not affect the expression of CCNE, thereby suggesting that CCNE overexpression was possibly induced by the phosphorylation of Rb proteins.miR-152 transfection and CDK5R1 knockdown (CDK5R1 siRNA-transfected) led to the downregulation of Rb phosphorylation and reduced expression of CCNE and CCNA.Reduced Rb phosphorylation, CCNE and CCNA expression after reduced expression of CDK5R1 were observed in the S phase as well.Interestingly, the expression of CCNE persisted in the G2/M phase, instead of being reduced.Throughout the cell cycle, phosphorylation of Rb proteins through CDK5 suggests the direct phosphorylation of Rb proteins mediated by CDK5.Further, we revealed miR-152 to be a tumor-suppressive miRNA that reduces the expression of p35 and inhibits the activation of CDK5-p25 complex in the nucleus of ES cells.The results indicate that a stable supply of CDK5 cleaved from CDK5R1 to the nucleus, regardless of the cell cycle phase, causes phosphorylation of Rb.This suggests that E2F1 permanently expresses its target genes, CCNE and CCNA, which is consistent with our results.
The finding that reduced expression of CDK5R1 led to tumor shrinkage in mice is consistent with previous findings on the cell proliferative effect of CDK5R1.It was shown that the transfection with miR-152 mimic reduced the expression of CDK5R1 and CCNE in tumor tissues, even in mice.Reduced expression of miR-152 that was observed in all ES cells allowed activation of CDK5 in the nucleus via the overexpression of p35 and p25, thereby indicating a relationship between the reduced expression of tumor suppressive miR-152 and continuous activation of CCNE through various factors.Previous studies have reported the overexpression of CCNE in cancer cells as well.
Our current findings on the activation of CDK5 induced by the overexpression of CDK5R1 and p25 are consistent with those reported mainly in neuronal studies.However, the effects of CDK5 have not been previously discussed in detail, and this study elucidated the mechanism by which abnormal control by miRNAs disrupted CCNE expression via cell cycle progression through Rb-related disinhibition.The relationship between www.nature.com/scientificreports/CDK5R1 and Rb phosphorylation and CCNE/CDK2 in ES has not been fully elucidated.However, CCNE has been identified as a predictor of therapeutic efficacy because its overexpression reduces sensitivity to CDK4/6 inhibitors 24 .Drugs targeting the cell cycle through Rb phosphorylation are currently in clinical use, and CCNE is the final effector molecule.Thus, our results demonstrate the utility of our approach for the development of effective individualized treatments for ES.

Ethical approval
Each author certifies that his or her institution has approved the animal protocol for this investigation and that all investigations were conducted in conformity with ethical principles of research.Mouse experiments were approved by the Medical Ethics Committee of Oita University (No. 182403) and all experiments were performed in accordance with relevant guidelines and regulations.All animal experimental procedures were performed in accordance with ARRIVE guidelines 25 .

Cell lines
ES cell lines, SKES-1, RDES, SKNMC, and SCCH were purchased from the Japanese Collection of Research Bioresources Cell Bank (Tokyo, Japan), and the WE68 cell line was kindly provided by Dr. Frans van Valen (Westfalische-Wilhelms University, Munster, Germany).Human mesenchymal stem cells (hMSCs) were obtained from TaKaRa Biotechnology (Otsu, Japan).All cell lines used are routinely (every 3 months) tested for Mycoplasma using the e-Myco PLUS Mycoplasma PCR Detection Kit (LiliF Diagnostics).RDES and SKNMC cells were cultured in Dulbecco's modified eagle medium (DMEM) high glucose medium (Invitrogen, NY) with 10% FBS and 1% penicilium and streptomycin.SKES1 cell and WE-68 cells were cultured in RPMI 1640 (Invitrogen, NY) supplemented with 10% FBS.SCCH cells were grown in minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS; Invitrogen, NY) and 0.1 mmol/L nonessential amino acids (NEAA).hMSCs were

Figure 1 .
Figure 1.CDK5R1 is the target gene of miR-152.(a) The binding site of CDK5R1 and miR-152 is shown.(b) The sequence of miR-152 and the sequence of miR-152 mutant with mutation in the seeded region.After transfection of SKES-1 cells, miR-152 was detected by quantitative PCR from RNA extracted from the cells.miR-152 was appropriately introduced into the cells, and the miR-152 mutant did not differ from the control group.(c) Intracellular expression of CDK5R1 after transfection with miR-152 or miR-152 mutant as analyzed by reverse transcriptase-quantitative polymerase chain reaction (qRT-PCR).(d) Predicted binding sites of miR-152 in the 3′-untranslated region (UTR) of CDK5R1 binding sites 1 and 2. Sites mutated as controls for the luciferase assay are underlined.WT, wild-type; M1, mutation of CDK5R1 3′-UTR binding site 1; M2, mutation of CDK5R1 3′-UTR binding site 2. (e) Luciferase assay identified CDK5R1 as two targets of miR-152.Luciferase reporter assay for the direct and specific interaction of miR-152 with predicted two target sites in the 3′-UTR of CDK5R1.Reporter activity was significantly reduced in miR-152 mimic transfected cells.Control-miR cells were used as control here for normalization.The mutations at the two target sites did not reduce luciferase activity in response to the miR-152 mimic; it remained equivalent to the Control-miR group.Images (f) and plots (g) from the immunoblot analysis of CDK5R1 protein expression following changes in miR-152 expression.Images (h) and plots (i) of protein expression of CDK5R1 following changes in the concentration of CDK5R1 siRNA.GAPDH level was used as loading control. https://doi.org/10.1038/s41598-023-45833-6

Figure 2 .
Figure2.Changes in the expression of p35/p25 in the cytoplasm and nucleus, and changes in the expression of CDK5R1 after transfection with miR-152.Effects of activation and inhibition of calpain on p35/p25, and subsequent changes in CDK5 activity in the cytoplasm and nuclear fractions were analyzed by immunoblotting (a) and protein expression quantification (b).The cytoplasmic fraction was normalized using GAPDH, and the nuclear fraction was normalized using Lamin B1 as reference.In the nuclear fraction, p25 was increased by calpain activation and p35 was absent, whereas nuclear p25 was decreased by calpain inhibitor.Cytoplasmic p25 was formed from the cleavage of p35 by calpain activation and inhibition.Effects of miR-152 mimic transfection and CDK5R1 knockdown on p35/p25 and CDK5 in the cytoplasm fractions were analyzed by western blot analysis (c) and band concentration quantification (d).Immunoprecipitation (IP) was performed with CDK-5, followed by western blotting with the indicated antibodies.The Input was normalized using GAPDH, and the proteins immunoprecipitated with CDK5 were normalized to CDK5.Western blotting analysis of p35/p25 levels in SKES-1 cells transfected with miR-152 and CDK5R1 siRNA in nucleus fraction (e).Immunoprecipitation (IP) was performed with CDK-5, followed by western blotting with the indicated antibodies.Quantification (f) of changes in the protein expression of co-precipitated p35/p25 in the nuclear fractions were performed.The Input of nucleus fraction was normalized using Lamin B1, and the proteins immunoprecipitated with CDK5 were normalized to CDK5.

Figure 3 .
Figure 3. Effects and causes of changes in miR-152 and CDK5R1 expression on cell proliferation.(a) Cell proliferation of SKES-1 and RDES cells transfected with miR-152 mimic or CDK5R1 siRNA were compared with those in the negative control group.Data represent mean ± SEM of three independent experiments.SKES-1 cells transfected negative control-miR (20 nM), miR-152 mimic (20 nM), negative control-siRNA (20 nM), CDK5R1-siRNA (20 nM) and untreated cells, followed by western blot analysis with the indicated antibodies.Images (b) from immunoblot analysis of apoptosis induction after transfection with miR-152 mimic and CDK5R1 siRNA.GAPDH level was used as loading control.(c) Ewing's sarcoma cells were stained with Annexin V-FITC/PI to assess cell apoptosis.Each quadrants represent viable cells (Lower Left quadrant), early apoptotic cells (Lower Right), late or secondary necrotic cells (Upper Right), and primary necrotic cells (Upper Left), respectively.(d) The viability of SKES-1 and RDES cells was determined using Cell Counting Kit-8 assays.(e)Cell proliferation was assessed by observing the changes in the expression of Ki-67 and PCNA using qPCR.SKES-1 cell-transfected negative control-miR (20 nM), miR-152 mimic (20 nM), negative control-siRNA (20 nM), CDK5R1-siRNA (20 nM) and untreated cells, followed by flow cytometric analysis with 5-bromo-2 deoxyuridine-7-amino-actinomycin D (BrdU-7-AAD) staining.Changes in cell cycle for each condition were analyzed by the BrdU-7-AAD double staining assay (Supplemental Fig.S3c) and the rate of cell-cycle progression (f).(g) Effect of miR-152 mimic and CDK5R1 knockdown on the viability of SKES-1 cells for their ability to grow in soft agar.The results are represented as the mean ± SEM (n.s., no significance).**p < 0.01 versus the related control groups.

Figure 4 .
Figure 4. Effects of cell cycle factors after transfection with oligonucleotides in the G1 phase.Negative control-miR (20 nM), miR-152 mimic (20 nM), negative-control siRNA (20 nM), and CDK5R1-siRNA (20 nM) were transfected in SKES-1 cells and were synchronized at different stages of the cell cycle: G1 phase, thymidine treatment (2 mM, 24 h).Nuclear protein was extracted, and western blotting was performed with the indicated antibodies.Phosphorylation of p-CDK2 (Thr160), p-CDC2 (Thr161), and p-Rb (Ser807/811) is inhibited by treatment with miR-152 mimic and CDK5R1 siRNA.Cell cycle factors after treatment were analyzed by immunoblotting (a) and band concentration quantification (b).The protein bands of Cell cycle-related proteins were quantified and normalized to Lamin B1.The results are represented as the mean ± SEM (n.s.no significance).**p < 0.01 versus the related control groups.C. Representative confocal images of Phosphorylated (p) -Rb are shown.p-Rb was stained red; whereas, F-actin was stained with phalloidin in green (c).Nuclear staining was performed with DAPI.Scale bar, 20 μm.

Figure 5 .
Figure 5. Effects of cell cycle factors after transfection with oligonucleotides in the S phase.Negative control-miR (20 nM), miR-152 mimic (20 nM), negative-control siRNA (20 nM), and CDK5R1-siRNA (20 nM) were transfected in SKES-1 cells and were synchronized at different stages of the cell cycle: S phase, hydroxyurea treatment (3 mM, 24 h).Nuclear protein was extracted, and western blotting was performed with the indicated antibodies.Cell cycle factors after treatment were analyzed by immunoblotting (a) and band concentration quantification (b).The protein bands of Cell cycle-related proteins were quantified and normalized to Lamin B1.The results are represented as the means ± SEM (n.s.no significance).**p < 0.01 versus the related control groups.C. Representative confocal images of p-Rb are shown.p-Rb was stained red, whereas F-actin was stained with phalloidin in green (c).Nuclear staining was performed with DAPI.Scale bar, 20 μm.

Figure 6 .
Figure 6.Effects of cell cycle factors after transfection with oligonucleotides in the G2/M phase.Negative control-miR (20 nM), miR-152 mimic (20 nM), negative-control siRNA (20 nM), and CDK5R1-siRNA (20 nM) were transfected in SKES-1 cells and were synchronized at different stages of the cell cycle: G2/M phase, nocodazole treatment (50 ng/mL, 24 h).Nuclear protein was extracted, and western blotting was performed with the indicated antibodies.Cell cycle factors after treatment were analyzed by immunoblotting (a) and band concentration quantification (b).The protein bands of Cell cycle-related proteins were quantified and normalized to Lamin B1.The results are represented as the mean ± SEM (n.s.no significance).**p < 0.01 versus the related control groups.(c) Representative confocal images of p-Rb are shown.p-Rb was stained red, whereas F-actin was stained with phalloidin in green.Nuclear staining was performed with DAPI.Scale bar, 20 μm.

Figure 7 .
Figure 7. Effects of miR-152 and CDK5R1 were examined in mice.SKES-1 cells (1 × 10 6 ) were inoculated into the gluteal region of BALB/c nu/nu mice.After 5 weeks, the mouse tumors were collected and analyzed for further experiments.Tumor volumes in implanted mice (a) were compared in each group (b).The subcutaneous tumors are indicated by red dashed lines.The collected mouse tumor tissues were analyzed by protein expression (c) and immunostaining of CDK5R1 and CCNE (d) per quantification unit area in CDK5R1-and CCNE-positive cells was quantified.The densitometric levels of protein bands were quantified and normalized to that of GAPDH.Original magnification, × 400; Scale bars: 50 μm.Data represent mean ± SEM of three independent experiments.**p < 0.01 versus the related control groups.(e) The schema illustrates that low expression of miR-152 causes accumulation of CDK5R1 and contributes to high expression of CCNE.